USP21-mediated G3BP1 stabilization accelerates proliferation and metastasis of esophageal squamous cell carcinoma via activating Wnt/β-Catenin signaling

Lacking effective therapeutic targets heavily restricts the improvement of clinical prognosis for patients diagnosed with esophageal squamous cell carcinoma (ESCC). Ubiquitin Specific Peptidase 21 (USP21) is dysregulated in plenty of human cancers, however, its potential function and relevant molecular mechanisms in ESCC malignant progression as well as its value in clinical translation remain largely unknown. Here, in vitro and in vivo experiments revealed that aberrant upregulation of USP21 accelerated the proliferation and metastasis of ESCC in a deubiquitinase-dependent manner. Mechanistically, we found that USP21 binds to, deubiquitinates, and stabilizes the G3BP Stress Granule Assembly Factor 1 (G3BP1) protein, which is required for USP21-mediated ESCC progression. Further molecular studies demonstrated that the USP21/G3BP1 axis played a tumor-promoting role in ESCC progression by activating the Wnt/β-Catenin signaling pathway. Additionally, disulfiram (DSF), an inhibitor against USP21 deubiquitylation activity, markedly abolished the USP21-mediated stability of G3BP1 protein and significantly displayed an anti-tumor effect on USP21-driving ESCC progression. Finally, the regulatory axis of USP21/G3BP1 was demonstrated to be aberrantly activated in ESCC tumor tissues and closely associated with advanced clinical stages and unfavorable prognoses, which provides a promising therapeutic strategy targeting USP21/G3BP1 axis for ESCC patients.


Fig. S6. USP21 depletion or overexpression alters G3BP1 protein levels but does not affect its mRNA expression.
A Western blot analysis was applied to determine the expression of G3BP1 protein in Eca-109 cells transfected with si-NC, si-USP21#1, or si-USP21#2.B G3BP1 mRNA levels were evaluated in KYSE-150 and Eca-109 cells with depletion of USP21 via RT-qPCR.C G3BP1 protein levels were assessed using a western blot in Eca-109 with ectopic expression of Vector, USP21 WT , or USP21 C221A .D RT-qPCR was conducted to measure G3BP1 mRNA levels in KYSE-150 and Eca-109 cells with USP21 overexpression.E Protein lysate was respectively collected from the cytoplasm or nucleus in Eca-109 cells expressing ectopic USP21 WT and then subjected to western blot for indicated proteins.GAPDH and Lamin B1 were used as an internal control for the cytoplasmic and nucleus protein lysate, respectively.Data are shown as mean values ± SD.An unpaired t-test was applied for the determination of statistical significance in (B, D).
Corresponding P-values are indicated on the graphs.

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Fig. S1 .
Fig. S1.USP21 protein levels in ESCC tumors with different differentiation, age, or gender.A-C The difference in USP21 IHC scores between different groups of differentiation (A), age (B), or gender (C).The Mann-Whitney test was used to evaluate statistical significance (A-C).

Fig. S2 .
Fig. S2.USP21 depletion inhibits the growth, migration, and invasion of ESCC cells.A The quantitation of western blots for the efficiency of USP21 depletion in KYSE-150 cells was performed.B, C Western blot analysis (B) and its quantitation (C) were applied to detect the levels of indicated proteins in Eca-109 cells with transfection of si-NC, si-USP21#1, or si-USP21#2.D KYSE-150 cells expressing Vector, USP21 SR , or USP21 WT were transfected with si-NC, si-USP21#1, or si-USP21#2, followed by western blot for Flag-tag.E, F Plate colony formation (E) and transwell assays (F) were used to evaluate the growth, migration, and invasion capability of Eca-109 cells with indicated transfection.Representative stainings and statistical quantification as indicated.The scale bar (red line) in (F) is 100 μm.The data are presented as means ± SD, and an unpaired t-test was used to identify the statistical significance in (A, C, E, F).

Fig. S5 .
Fig. S5.IHC analysis of USP21 and Ki-67 proteins and H&E staining of pulmonary metastatic nodules in xenograft models derived from KYSE-150 cells with the expression of Vector, USP21 WT , or USP21 C221A .A The protein levels of USP21 and Ki-67 were detected in tumor sections of subcutaneous xenograft mice models with KYSE-150 expressing Vector, USP21 WT , or USP21 C221A .B Representative H&E staining images from pulmonary metastatic nodules of mice injected with Vector-, USP21 WT -, or USP21 C221A -expressing KYSE-150 cells.Scale bars as shown on their respective panels.

Fig. S8 .
Fig. S8.G3BP1 is essential for USP21-mediated ESCC progression.A Western blot analysis was used to detect indicated protein levels in Vector-or USP21 WT -expressing Eca-109 cells transfected with si-NC or si-G3BP1.B-D Plate colony formation (B) and transwell assays (C, D) were applied to evaluate the growth, migration, and invasion of Eca-109 cells with the expression of Vector + si-NC, USP21 WT + si-NC, Vector + si-G3BP1, or USP21 WT + si-G3BP1.Representative staining and statistical quantification as shown.E Western blot analysis was performed to determine indicated protein levels in Vector-or G3BP1 WT -expressing Eca-109 cells transfected with si-NC or si-USP21#1.F-H Plate colony formation (F) and transwell assays (G, H) were performed to assess the growth, migration, and invasion of Eca-109 cells expressing si-NC + Vector, si-NC + G3BP1 WT , si-USP21#1 + Vector, or si-USP21#1 + G3BP1 WT .Representative images and statistical quantification as indicated.Scale bars (red line) are 100 μm in (C, D, G, H).The data are presented as means ± SD and the unpaired t-test was used to determine the statistical significance (B-D, F-H).All P-values are indicated on the corresponding graphs.

Fig. S9 .
Fig. S9.USP21 regulates β-Catenin protein levels through G3BP1.A, B The protein levels of β-Catenin were detected using western blot in Eca-109 cells with USP21 knockdown (A) or overexpression (B).C, D The cytoplasmic and nuclear proteins were extracted separately from KYSE-150 transfected with si-NC, si-USP21#1 or si-USP21#2 (C) or expressing Vector, USP21 WT , or USP21 C221A (D), followed by western blot detecting β-Catenin levels.E The quantitation of western blots was from three replicate experiments with indicated information.F KYSE-150 cells were transfected with si-NC + Vector, si-NC + G3BP1 WT , si-USP21#1+Vector, or si-USP21#1+G3BP1 WT followed by western blot for β-Catenin protein.G, H Western blots from indicated experiments repeated three times were quantified.The data are displayed as means ± SD.The statistical significance (E, G, H) was identified using the unpaired t-test, and all P-values are shown on the corresponding graphs.

Fig. S11 .
Fig. S11.The clinical significance of dysregulated G3BP1 protein expression.A The ROC analysis was applied to assess the diagnostic value of G3BP1 protein expression for ESCC.B-G IHC scores of G3BP1 in ESCC tissues were compared among patients with different T statuses of primary tumor (B), with N0 or N1-3 statuses of regional lymph nodes (C), with different ESCC stages (D), or with different groups of differentiation (E), age (F), gender (G).H, I OS (H) and PFS (I) in ESCC patients with high or low G3BP1 protein levels were analyzed using Kaplan-Meier curves.The P-values were determined using a Mann-Whitney test in (B-G) and a log-rank test in (H, I).All P-values and n numbers as indicated.